Studies on replication of plasmid Col E1 DNA have been continued. Nucleotide sequence of the region around the origin of replication has been determined. The region of about 600 base pairs was found to be essential for replication and maintenance of the plasmid. Initiation of replication from the specific region can be initiated with RNA polymerase, RNase H and DNA polymerase I. A hybrid plasmid containing both origins of replication of Escherichia coli chromosome and plasmid Col E1 was isolated.